Tetracycline activity enhancement using doxycycline or sancycline

ABSTRACT

Methods and products for overcoming bacterial resistance to tetracycline-type antibiotics by inhibiting the plasmid-mediated active efflux system for tetracycline in the resistant bacterial cell by administering with tetracycline efflux system blocking agents of doxycycline or sancycline, thereby increasing the sensitivity of the resistant cell to tetracycline type antibiotics.

This is a continuation of copending application Ser. No. 06/850,843filed on Apr. 11, 1986, now U.S. Pat. No. 5,021,407, which is acontinuation of U.S. Ser. No. 06/442,688, filed Nov. 18, 1982 now U.S.Pat. No. 4,806,529.

FIELD OF THE INVENTION

This invention relates to methods and products for enhancing theeffectiveness of antibiotics, more particularly, to methods and productsfor overcoming bacterial resistance to antibiotics of the tetracyclinefamily.

BACKGROUND OF THE INVENTION

The tetracyclines are bacteriostatic antibiotics used to treat a broadspectrum of microbial disease agents in humans, animals and plants.

Many bacteria are able to adapt to their environment in ways whichpermit them to become resistant to antibiotics. Strains of group Astreptococci devoloped resistance to sulfadiazine during World War II.Resistant staphylococcal infections began to spread through publicinstitutions and hospitals following the widespread use of penicillin.Since the introduction of tetracyclines into clinical practice, a numberof microorganisms have developed resistance to these drugs. Treatment ofbacterial infections in palm trees with tetracycline solutions throughthe root system has become increasingly ineffective. See Levy, "TheTetracyclines: Microbial Sensitivity and Resistance," New Trends inAntibiotics: Research and Therapy, Elsevier/North-Holland BiomedicalPress, 1981, pp. 27-44; Chopra et al., "The tetracyclines: prospects atthe beginning of the 1980's," Journal of Antimicrobial Chemotherapy,8:5-21, (1981,) which are incorporated herein by reference.

Tetracycline was one of the real wonder drugs when introduced into theclinical world in 1948. However, many microorganisms have developedresistance to tetracycline. More alarming is the emergence of organismswith resistance to the newer tetracycline analogs. Resistant bacteriaare also appearing among individuals who have not consciously ingestedthe drug. It has therefore become increasingly important to determinethe mode of resistance and to develop a method of circumvention. Sincethe mechanism does not degrade the drug and breakdown in nature issmall, tetracycline remains in the environment to continue to promoteemergence of resistant organisms. Thus determination of a method thatwould overcome this resistance would provide a substantial increase inthe effectiveness of tetracyclines, while reducing the present largeincrease in tetracycline resistant microbial diseases.

Investigations into the mode of action of the tetracyclines supportobservations that the tetracyclines inhibit the protein synthesis ofsensitive bacteria at the level of the ribosome, as described inLehninger, Biochemistry--The Molecular Basis of Cell Structure andFunction, (2d ed., Worth Publishers, 1975), p. 941, which isincorporated herein by reference. This inhibition interferes with totalprotein synthesis and biosynthesis of the bacterial respiratory system.

In the resistant organism, resistance does not promote inactivation ofthe tetracycline molecule. Rather, the total efflux rate is increasedand the steady state accumulation by the cell is obtained at a lower,biologically ineffective concentration of drug.

Resistance to the tetracyclines in most bacterial species is specifiedby extra-chromosomal, autonomously replicating and often transmissibleplasmids, called R factors, which carry genes which mediate tetracyclineresistance. Two kinds of tetracycline resistance determinants amongplasmids have been described: those with resistance to tetracyclinealone and those with resistance to tetracycline and its lipophilicanalogs. It has been shown that at least four different genetic elementsencode the tetracycline resistance phenotype. See Mendez et al.,"Heterogeneity of Tetracycline Resistance Determinants," Plasmid,3:99-108, 1980, which is incorporated herein by reference.

Plasmid mediated resistance is inducible in many bacteria. Theresistance level can be experimentally increased by preincubation of thecells in subinhibitory amounts of tetracycline. It was found thatcoincident with induced resistance was the induced synthesis of aplasmid-encoded inner membrane protein, which was designated "TET"protein. See Levy and McMurry, "Detection of an Inducible MembraneProtein Associated with R-Factor-Mediated Tetracycline Resistance."Biochemical and Biophysical Research Communications, 56(4):1060-68,(1974), which is incorporated herein by reference. Moreover,accumulation of drug by resistant cells was dramatically different fromthat accumulation in sensitive cells. See Levy and McMurry,"Plasmid-determined Tetracycline Resistance involves new transportsystems for tetracycline, " Nature, 275 (5683): 90-92 (1978), which isincorporated herein by reference. While tetracycline was activelyaccumulated by sensitive cells (McMurray and Levy in "Two transportsystems for Tetracycline in Sensitive Escherichia coli: Critical rolefor an Initial Rapid Uptake System Insensitive to Energy Inhibitors,"Antimicrobial Agents and Chemotherapy 14(2); 201-09 (1978), which isincorporated herein by reference), these uptake systems were found to bealtered by at least one tetracycline resistance plasmid, R222. Nature275 (5683), supra, at 91. They subsequently demonstrated that all fourplasmid-borne tetracycline resistance determinants specified an activeefflux system for tetracycline, (McMurray et al., "Active Efflux ofTetracycline Encoded by Four Genetically Different TetracyclineResistance Determinants in Escherichia coli," Pro. Nat. Acad. of Sci.USA 77 (7): 3974-77 (1980), which is incorporated herein by reference).More recently, using tetracycline sensitive mutations which mapped inthe TET structural region, the investigators demonstrated twogenetic-complementation groups designated TET A and TET B. Absence ofeither one of these gene loci causes loss of the energy-dependent eflluxof tetracycline which is characteristic of tetracycline resistance.(Curiale and Levy, "Two Complementation Groups Mediate TetracyclineResistance Determined by Tn10," Journal of Bacteriology, 151(1): 209-15(1982), which is incorporated herein by reference.)

It is among the objects of the present invention to provide a processfor enhancing the bacteriostatic and bacteriocidal effects of thetetracyclines. It is also an object to provide a process forcircumventing the tetracycline efflux mechanism of resistant bacterialcells. It is also an object to provide a process for promotingaccumulation of minimum inhibitory concentrations of tetracyclineswithin the bacterial cell. It is also an object to provide a process forconverting a tetracycline resistant cell into a tetracycline sensitivecell. Additional objects and advantages of the invention will be setforth in part in the description which follows, and in part will beobvious from the description, or may be learned by practice of theinvention. The objects and advantages of the invention may be realizedand obtained by means of the instrumentalities and combinationsparticularly pointed out in the appended claims.

SUMMARY OF THE INVENTION

The invention herein comprises methods and products for inhibition ofthe function of the plasmid-mediated active efflux system fortetracycline-type antibiotics in the bacterial cell, whereintetracycline-type antibiotics are administered to the cell incombination with tetracycline efflux system inhibitory or blockingagents. The active efflux system in resistant cells is thus inhibited,and the tetracycline type antibiotic is effective at lower concentrationto terminate cellular protein synthesis in previouslytetracycline-resistant microorganisms. The use of such combinationsprovides a process for enhancing the bacteriocidal and bacteriostaticeffects of the tetracyclines. When the efflux system of the resistantcell is blocked, the resistant cell converts to sensitive cellcharacteristics.

DETAILED DESCRIPTION OF THE INVENTION

In accord with the present invention, a method of blocking thetetracycline efflux system of tetracycline resistant cells is obtainedby treating tetracycline resistant cells with an efflux blocking agent,i.e., a tetracycline analog or another type of agent which interfereswith the action of the TET A and/or TET B proteins or protein domainsand thus decreases efflux of tetracyclines from the cell. This methodrenders formerly resistant cells non-resistant, i.e., sensitive totetracycline. Inhibition of the tetracycline efflux system may bedemonstrated by a comparison of the transport of tetracycline with andwithout an efflux blocking agent by susceptible cells and by resistantcells. Preferably, tetracycline type antibiotics are administered inconjunction with, or shortly after treatment with the efflux blockingagent.

By "tetracycline-type antibiotic", tetracyclines, or the tetracyclinefamily, as used herein, is meant tetracycline and its analogs, which arecompounds having the structural formula: ##STR1## wherein R₁ to R₅ maybe hydrogen, hydroxy, alkyl, substituted alkoxy, alkylene, halogen, etc.See Korolkovas et al., Essentials of Medicinal Chemistry (John Wiley &Son, Inc., 1976), at 512-17, the disclosure of which is incorporatedherein by reference. Preferably R₁ and R₂ are hydrogen or hydroxy, R₃ ishydrogen or methyl, R₄ is hydrogen, halogen, preferably chlorine, oramino, preferably dimethylamino, and R₅ is hydrogen, N-methyl pyrole,(as in rolitetracycline) or ##STR2## (as in lymecycline). R₂ and R₃together may be methylene, as in methacycline.

In tetracyline sensitive cells, tetracycline is accumulated so that theintracellular concentration of tetracyclines exceeds the extracellularlevel. Part of the uptake occurs via an initial energy-independent phaseof antibiotic uptake as detailed in Antimicrobial Agents 14 (2), supraat 201. However, at least half of the uptake occurs via an energydependent system sensitive to metabolic inhibitors.

In resistant organisms, an additional energy-demanding andcarrier-mediated active efflux system is incorporated by the presence ofresistance-determinator genetic material, such as a TET resistant geneencoded by a plasmid. Resistance does not occur by means of inactivationof the tetracycline molecule. Rather the total efflux rate is increased,so that, at a steady state, the resistant organism has a lowintracellular level of tetracycline. This resistance mechanism is anactive system for pumping the drug out of the cell.

We have now found that efflux of tetracycline type antibiotics can beblocked by use of a blocking agent which binds, associates with, orotherwise deactivates the carrier protein(s) which are active ineffluxing tetracyclines from the cell. While not wishing to be bound bytheory, it is believed that the proteins or protein domains known as TETA and TET B, or other similar carrier proteins, e.g. from othertetracycline resistance determinants, actively instill resistance inmicroorganisms by binding to or otherwise associating with thetetracycline type antibiotic and transporting the antibiotic out of thecell. It further appears that both TET A and TET B proteins or proteindomains have a critical and supportive role in affecting the effluxsystem, and thus it is possible to block the efflux system and convertresistant microorganisms to sensitive microorganisms, by use of ablocking agent which binds or associates with either TET A and/or TET Bproteins.

In accord with the present invention, the preferred blocking agents areanalogs or derivatives of tetracyclines, or compounds which contain asufficient part of the tetracycline structure such that they arerecognized by and bound to or otherwise associated with at least one ofthe carrier protein molecules or domains which are responsible foreffluxing tetracycline-type antibiotics, and thus are efficient indisrupting the efflux system of the microorganism involved. Suitableblocking agents include but are not limited to the known tetracyclineantibiotics, including oxytetracycline, chlorotetracycline,demeclocycline, doxycycline, B-chelocardin, minocycline,rolitetracycline, lymecycline, sancycline and methacycline, and othercompounds including latent forms of tetracycline, such as apicycline,clomocycline, guamecycline, meglucycline; mepycycline, penimepicycline,pipacycline, etamocycline, penimocycline, etc. Tetracycline(s) may alsobe used in salt form, e.g. as a tetracycline lactate, t. lauryl sulfate,t. phosphate complex, t. cyclohexyl sulfamate, or other pharmaceuticallyacceptable salts.

The amount of blocking agent to be used varies with the efficiency ofits blocking activity, its absorption by the organism being treated, andthe degree of resistance of the microorganism. Sufficient amounts of theblocking agent should be used to make the microorganism susceptible to apharmaceutically acceptable level of tetracycline in the man, animal orplant being treated. The molar ratio of blocking agent to tetracyclineor tetracycline type antibiotic which is administered may generally befrom 0.01 to 100, preferably from 0.05 to 2.0 and more preferably 0.05to 1.0. In in vivo treatment, the blocking agent may be administered inamounts which are sufficient to exhibit blocking effect, but which donot adversely affect the subject. This does not apply to use of thisinvention in vitro, e.g., in processing chemical reactions, etc.Generally, the daily dosage of blocking agent for treatment of diseasein mammals may range from 0.01 to 100 mg/Kg normal body weight,preferably in an amount of about 0.1 to 50 mg/Kg body weight. Theblocking agent may be administered separately from the tetracycline typeantibiotic, but preferably is administered simultaneously with thetetracycline type antibiotic. Typically, tetracycline-type antibioticswill be administered in a regular daily course of treatment, to attainand maintain a concentration in the blood or the bodily fluids whichwill inhibit the microorganism being treated. Since the presence of theblocking agent deactivates the resistance of the microorganism to thetetracycline type antibiotic, the blocking agent should also be utilizedin a continuing treatment to render the antibiotic treatment effective.

Non-tetracycline based compounds may be utilized as blocking agents,provided that their structure is such as to interact with the carrierswhich cause antibiotic efflux so as to prevent or decrease that efflux.The efficiency of blocking agents in reducing efflux of tetracyclinetype antibiotics can be determined by testing against a tetracyclineresistant bacteria. The bacteria used may be naturally occurringtetracycline resistant bacteria, or may be made by incorporatingplasmids which code for tetracycline resistance into other bacteriahosts.

Preferably the blocking agent and a tetracycline type antibiotic arecombined in a pharmaceutical composition with a pharmaceuticallyacceptable carrier. The active ingredients may be administered by anyroute appropriate to the condition to be treated, suitable routesincluding oral, nasal (e.g., by spray) and parenteral (includingsubcutaneous, intramuscular and intravenous). It will be appreciatedthat the preferred route will vary with the condition to be treated.

While it is possible for the blocking agent to be administered as theraw chemical, it is preferable to present it as a pharmaceuticalformulation preparation.

The formulations, for veterinary, agricultural and human use, of thepresent invention comprise the active ingredient, e.g. blocking agentplus tetracycline family drug, as above defined, together with one ormore acceptable carriers therefore and optionally other therapeuticingredients. The carrier(s) must be "acceptable" in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. Desirably the formulation shouldnot include oxidizing agents and other substances with which theseantibiotics and their derivatives and blocking agents are known to beincompatible. The formulations include those suitable for oral orparenteral (including subcutaneous, intramuscular and intravenous)administration, although the most suitable route in any given case willdepend upon for example, the active ingredient and the condition to betreated. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing into association theactive ingredient with the carrier which constitutes one or moreaccessory ingredients. In general the formulations are prepared byuniformly and intimately bringing into association the activeingredients with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product into the desiredformulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; or as a solution or a suspension in an aqueousliquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, nasal spray, suppository, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent.

Formulations suitable for parenteral administration convenientlycomprise sterile aqueous solutions of the active ingredient whichsolutions are preferably isotonic with the blood of the recipient. Suchformulations may be conveniently prepared by dissolving the activeingredient in water to produce an aqueous solution, and rendering saidsolution sterile. These formulations may be presented in unit ormulti-dose containers, for example sealed ampoules or vials.

It should be understood that in addition to the aforementionedingredients the formulations of this invention may include one or moreadditional ingredients such as diluents, buffers, flavouring agents,binders, surface active agents, thickeners, lubricants, preservatives(including anti-oxidants) and the like.

Where the formulation, for human or for veterinary use, is presented inunit dosage form, for example those unit dosage forms specificallymentioned above, each unit thereof conveniently contains the activeingredient (as above defined) in an amount in the range of about 1 mg toabout 1000 mg.

To review, a sensitive cell is constantly accumulating tetracycline viaan active uptake system and a passive diffusion mechanism. In theresistant cell, there is an active efflux in addition to the active andpassive uptake of the drug. The present invention overcomes the problemsassociated with the plasmid mediated tetracycline resistance by blockingthe efflux system with an inhibitor administered in combination withtetracycline analogs. It has been discovered that by blocking the activeefflux, the resistant cell accumulates the tetracycline analog just thesame as does the sensitive cell.

The present invention teaches that if the efflux system of the resistantcell is blocked, the resistant cell reverts to sensitive cellcharacteristics. Since the TET proteins are an expression of resistancemediated by the plasmid, blocking the action of the TET A and/or Bprotein or protein domain(s) will block the action of the efflux systemthereby rendering the resistant cell sensitive to tetracycline analogsonce again.

The effectiveness of a particular blocking agent can simply bedetermined by testing for minimum inhibitory concentration (MIC) of thetetracycline type antibiotic of choice, and comparing the MIC of thatantibiotic alone, with its MIC when used in combination with theblocking agent. The MIC of the antibiotic or antibiotic blocking agentcombination can be determined for example by following the procedureoutlined in Antimicrobial Agents, 14(2) supra at 202. Other methods ofmeasuring MIC's are well known in the art.

Uptake and/or efflux of tetracycline-type antibiotics or other agentsmay also be directly measured by counting the amount of radiolabeleddrug in whole cells. The advantage of the whole cell method is thattransport system saturation can be directly demonstrated. There is apoint where the amount of tetracycline influx is greater than simplediffusion or the amount of tetracycline lost via active efflux.

Another method of assaying the effectiveness of blocking agents is thevesicle method, which utilizes everted inner membrane vesicles. Pro.Nat. Acad. Sci. USA, 77(7), supra at 3974-5; McMurry et al., "ActiveUptake of Tetracycline by Membrane Vesicles from Susceptible Escherichiacoli, Antimicrobial Agents and Chemotherapy 20(3): 307-13 (1981), whichare incorporated herein by reference. Using the everted vesicles, thenormal efflux system may be observed as an influx system. The vesiclemethod has the advantage of measuring competition or binding with theefflux system when increasing amounts of tetracycline-type analogs orother agents are employed.

The following examples are set forth to further illustrate the presentinvention.

EXAMPLE I Transport of Minocycline in Susceptible and ResistantEscherichia coli.

The invention was demonstrated by use of minocycline, a semisysntheticanalog of tetracycline, to interfere with the efflux system of aresistant cell, allowing the formerly tetracycline resistant cell toaccumulate tetracycline actively. This example demonstrates that bothanalogs were effluxed by the same carrier in resistant cells and that bysaturating the efflux system with the analog minocycline, tetracyclinenet efflux from the resistant cell was stopped. The significance of thisobservation is combined with the further observation that addition ofthe efflux blocking agent does not affect the active uptake system fortetracycline. Thus, tetracycline continues to accumulate in theresistant cell. The presence of a blocking agent reduces the necessaryextracellular concentration of tetracycline to achieve a MIC.

Minocycline is a semisynthetic analog of tetracycline and is much morelipophilic than tetracycline. Plasmids which specify resistance totetracycline offer much less resistance to its more lipophilic analogminocycline. The level of minocycline resistance is generally 1% to 10%that of tetracycline.

Plasmid R222 contains the class B tetracycline resistance determinant onTn10. Minocycline resistance of R222 is only 6% of the tetracyclineresistance level. Minocycline resistance for another R plasmid, pIP7,which bears the class A tetracycline resistance determinant, is only 1%of the tetracycline resistance level. These plasmids were utilized tocompare the transport of the two tetracyclines by susceptible and by twodifferent resistant cells.

In sensitive cells, at low levels of drugs, net actively-accumulatedminocycline was about 60 times the external concentration; fortetracycline, the value was 7-8 times the external concentration.

Steady state accumulation of labeled tetracyclines was measured (30 minafter addition of label) in resistant cells as a function of externaldrug concentration in the presence and absence of dinitrophenol. Theseexperiments were identical to those described by McMurry and Levy,supra, Antimicrobial Agents and Chemotherapy 14(2) for susceptiblecells. Net active efflux was declared if steady-state uptake in thewhole cells in the presence of the energy inhibitor DNP was greater thanthat in its absence.

Normally, addition of energy inhibitors such as DNP or cyanide toresistant cells causes an increase in steady state tetracycline levels,Nature, supra, at 90, since active efflux is inhibited in thesedeenergized cells, Proc., supra at 3974. However, if this efflux weresaturated, accumulation in energized cells would no longer be lower. Infact, if resistant cells retained the active uptake system of the hostcell (which is unsaturable), this active uptake system might becomedetectable at external drug levels when the efflux system had beensaturated.

Net efflux of tetracycline in cells bearing plasmid R222 was unimpairedeven at external tetracycline levels of 1000 uM. The findings weredifferent with minocycline. While an efflux of minocycline was seen atconcentrations less than 6-7 uM, an active uptake of minocycline wasclearly revealed above this level. Above 20 uM the active uptake was 100times the external concentration, nearly equal to the 200 fold factorfor susceptible cells.

In contrast to these results with R222, cells harboring pIP7, which hada three-fold lower resistance to tetracycline and a twenty-fold lowerresistance to minocycline, showed that active tetracycline efflux incells disappeared at about 5 uM of tetracycline. As the external levelincreased, active uptake appeared. However, the amount of tetracyclinewithin the cells remained below that of susceptible cells at the sameexternal concentration (indicating that the tetracycline efflux was notyet saturated) until an abrupt step-up which occurred between 250 and400 uM. Active efflux of minocycline in these cells was only below 0.6uM. Above this level an active uptake of minocycline was demonstrated.

These results demonstrated that the host-mediated active uptake systemfor the tetracyclines was retained in resistant cells bearing eithertype of resistance determinant.

To ascertain whether each of the tetracyclines would interfere with theefflux system of the other, steady-state accumulation was measured oflabeled tetracycline in the presence of unlabeled minocycline and viceversa. Cells bearing R222 were used. First, various concentrations ofunlabeled minocycline were added with 3.4 uM [³ H] tetracycline. Atabout 10 uM unlabeled minocycline, the efflux disappeared, and at higherminocycline levels an active tetracycline uptake was seen. At 200 uMunlabeled minocycline, the highest concentration tested, the in/outratio (ratio of internal to external concentration) of energized cellswas 50, and of deenergized cells 5. This demonstrated that minocyclinewas interfering with the tetracycline efflux system.

At about 100 uM unlabeled tetracycline, efflux of [¹⁴ C] minocycline (at1.8 uM) disappeared and an active uptake appeared, indicating thatunlabeled tetracycline could also block the active efflux ofminocycline. At 400 uM tetracycline, the highest concentration tested,the in/out ratio of [¹⁴ C] minocycline in energized vs. deenergizedcells was 30 and 15 respectively, so the minocycline efflux system hadonly begun to saturate.

Thus, the efflux of both analogs probably occurred via the samesaturable carrier since each analog antagonized the efflux of the other.This finding was further verified by temperature sensitive efflux ofboth drugs in cells bearing a temperature sensitive tetracyclineresistance determinant on R222.

This example has demonstrated that tetracycline and minocycline areaccumulated in susceptible cells by both energy-independent andenergy-dependent uptake systems. This host-mediated energy-dependentuptake of both analogs was still present in tetracycline-resistantcells. The plasmid-mediated active efflux system previously describedfor tetracycline also effluxed the more lipophilic analog, minocycline,in resistant cells.

EXAMPLE II

In a similar manner competition experiments have been performed whichhave demonstrated that another tetracycline analog, chlortetracycline,effectively blocked efflux of minocycline via a cryptic efflux systemnewly discovered in sensitive E. coli cells.

EXAMPLE III

This example demonstrates that the minimum inhibitory concentration oftetracycline could be significantly reduced by the addition ofsubinhibitory levels of the tetracycline analogs.

In this example, minocycline hydrochloride (received from LederleLaboratories, N.Y. and described above) and thiatetracycline (receivedfrom E. Merck of Darmstadt, Germany), an analog of tetracycline, wereemployed. The level of thiatetracycline resistance in resistant cells isgenerally 1% that of tetracycline.

E. coli strain D1-209 (described in Proc. Natl. Acad. Sci. 77(7), supraat 3974) was employed for this example. Cells used in the uptakeexperiments were grown from A₅₃₀ =0.1 to A₅₃₀ =0.8 at 37° C. in Medium Awith 0.5% glycerol as previously described. Plasmid bearing cells wereinduced with 4uM tetracycline during growth. Cells were washed aspreviously described. The optical density was then reduced to 10⁻⁵ bydilution of the medium.

Subsequently, one ml of the dilute cellular stock solution was added totubes containing various analogs and concentrations. The lowestconcentration of antibiotic which prevented turbidity starting from aninitial inoculum at A₅₃₀ =10⁻⁵ was designated the minimal inhibitoryconcentration. The antibiotic concentrations chosen increased inincrements of approximately 15% of the magnitude of an initialapproximate MIC. Increments began at about 20% of the MIC and terminatedat about 200%.

Fresh solutions of drugs at 5 mM for minocycline and thiatetracyclineand at 40 mM for tetracycline were prepared weekly and stored at -15° C.Thiatetracycline was dissolved in ethanol. Other chemicals were utilizedas described in Example I.

One ml of the stock solution was added to each tube containing variouslevels of tetracycline to give the final concentrations. The MIC's forthe stock solutions at 17.5 hours were 480 uM for tetracycline, 20 uMfor minocycline, and 3.5 uM for thiatetracycline. Addition of 4 uM ofminocycline to the tetracycline stock solution produced an MIC fortetracycline at 400 uM. Addition of 8 uM of minocycline reduced the MICof tetracycline to 320 uM. Addition of 0.2 uM thiatetracycline into thetetracycline stock solution did not produce a change from thetetracycline MIC. However, addition of 0.8 uM of thiatetracyclinereduced the tetracycline MIC to 300 uM. Therefore, Example III hasconfirmed the results of Example I by using a subinhibitoryconcentration of another tetracycline analog in combination withtetracycline.

Thus, it has been demonstrated that the plasmid-mediatedtetracycline-resistance efflux system transports more than one kind oftetracycline. Different tetracycline analogs have been demonstrated tohave been transported at different rates in Example I. Sincetetracycline is being constantly accumulated within the sensitive orresistant cell, it is only necessary to block the efflux system.Similarly, addition of blocking agents such as minocycline andthiatetracycline greatly enhances the effectiveness of tetracyclines onpreviously resistant microorganisms.

These results are in accord with the scope of this invention, whichteaches that if tetracycline-type analogs or other products areadministered in combination with tetracycline to tetracycline-resistantcells, the analogs or other factors effectively inhibit the efflux oftetracycline allowing normal accumulation of tetracycline within thecell. In addition, lower concentrations of tetracycline were needed tokill resistant cells when tetracycline was administered in combinationwith tetracycline analogs. Thus, if the efflux system of the resistantcell is blocked, the resistant cell reverts to sensitive cellcharacteristics.

I claim:
 1. A pharmaceutical composition comprising:(a) a subinhibitoryamount of a blocking agent selected from the group consisting ofdoxycycline and sancycline; (b) tetracycline; and (c) a pharmaceuticallyacceptable carrier, wherein the blocking agent is employed in an amountwhich is sufficient to make the bacteria susceptible to apharmaceutically acceptable amount of tetracycline, the blocking agentcomponent of the composition being employed in a molar ratio of blockingagent to tetracycline of from about 0.01 to
 100. 2. The pharmaceuticalpreparation of claim 1, the blocking agent being employed in an amountof from 0.1 to 100 mg/kg body weight, and the molar ratio of blockingagent to the antibiotic being from about 0.05 to
 2. 3. A method ofovercoming the resistance of tetracycline resistant bacteria comprisingcontacting the bacteria with a composition consisting essentially of:(a)a subinhibitory amount of a blocking agent selected from the groupconsisting of doxycycline and sancycline; (b) tetracycline; and (c) apharmaceutically acceptable carrier, wherein the blocking agent isemployed in an amount which is sufficient to make the bacteriasusceptible to a pharmaceutically acceptable amount of tetracycline, andwherein the blocking agent is employed in a molar ratio of blockingagent to tetracycline from about 0.01 to 100.